Optimal control of Type I diabetes mellitus (DM) often requires a combination of rapid- acting and long-acting insulin analogs to achieve tight glycemic control. Current rapid- acting analogs, a pioneering triumph of biotechnology 15 years ago, are nonetheless too delayed in absorption for either ideal meal-time glycemic control or the safe and effective use of automated insulin pumps. We therefore seek to develop a second-generation rapid-acting insulin analog with substantially improved properties in order to provide additional benefits to patients. To this end, we first propose to optimize and apply a novel total chemical synthesis of insulin that provides for highly efficient folding/formation of disulfides. Facile chemical synthesis of insulin will enable the incorporation of a wide range of non-coded amino acids in order to systematically tune the properties of the insulin molecule. We will prepare a series of designed chemical analogs of insulin for biophysical characterization, receptor binding assays, and biological testing in animal models of DM. Our design goals for a second-generation insulin analog include: (i) faster onset of action; (ii) briefer duration of action; (iii) enhanced physical stability; (iv) enhanced chemical stability; and (v) enhanced receptor selectivity. Our proposed research promises to expand the chemical space of insulin therapeutics to exploit for the first time the armamentarium of modern medicinal chemistry.